Cartilage is a structural tissue constructed from a variety of hydrated biopolymers. By weight, water comprises 70% to 80% of cartilage. The remaining 20% to 30% comprises extracellular biopolymers such as collagen (primarily type-II) and proteoglycan. The collagen usually accounts for 70% of the dry weight of cartilage (in “Pathology” (1988) Eds. Rubin & Farber, J. B. Lippincott Company, PA. pp. 1369–1371). Proteoglycans are composed of a central protein core from which long chains of polysaccharides extend. These polysaccharides, called glycosaminoglycans, include: chondroitin-4-sulfate; chondroitin-6-sulfate; and keratan sulfate. Chondrocytes are cells responsible for the ordered production and secretion of the cartilagenous polymers. The properties of cartilage are primarily determined by the quantity and quality of the extracellular biopolymers.
Three types of cartilage are present in mammals and include: hyaline cartilage, fibrocartilage, and elastic cartilage (Rubin and Farber, supra). Hyaline cartilage consists of a gristly mass having a firm, elastic consistency, is translucent and is pearly blue in color. Hyaline cartilage is predominantly found on the articulating surfaces of articulating joints. It is found also in epiphyseal plates, costal cartilage, tracheal cartilage, bronchial cartilage and nasal cartilage. Fibrocartilage is essentially the same as hyaline cartilage except that it contains fibrils of type I collagen that add tensile strength to the cartilage. The collagenous fibers are arranged in bundles, with the cartilage cells located between the bundles. Fibrocartilage is found commonly in the anulus fibrosus of the invertebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis, and insertions of joint capsules. Elastic cartilage also is similar to hyaline cartilage except that it contains fibers of elastin. It is more opaque than hyaline cartilage and is more flexible and pliant. These characteristics are defined in part by the elastic fibers embedded in the cartilage matrix. Typically, elastic cartilage is present in the pinna of the ears, the epiglottis, and the larynx.
The surfaces of articulating bones in mammalian joints are covered with articular cartilage. The articular cartilage prevents direct contact of the opposing bone surfaces and permits the near frictionless movement of the articulating bones relative to one another (Clemente, supra).
Two types of articular cartilage defects are commonly observed in mammals and include full-thickness and partial-thickness defects. The two types of defects differ not only in the extent of physical damage but also in the nature of repair response each type of lesion elicits.
Full-thickness articular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified layer of cartilage located between the articular cartilage and the subchondral bone. Full-thickness defects typically arise during severe trauma of the joint or during the late stages of degenerative joint diseases, for example, during osteoarthritis. Since the subchondral bone tissue is both innervated and vascularized, damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of fibrocartilage at the site of the full-thickness defect. Fibrocartilage, however, lacks the biomechanical properties of articular cartilage and fails to persist in the joint on a long term basis.
Partial-thickness articular cartilage defects are restricted to the cartilage tissue itself. These defects usually include fissures or clefts in the articulating surface of the cartilage. Partial-thickness defects are caused by mechanical arrangements of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial-thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full-thickness defects.
Repair of articular cartilage defects with suspensions of isolated chondrocytes has been attempted in a variety of animal models. See for example: Bentley, et al. (1971) Nature 230:385–388; Langer et al. (1974) J. Bone Joint Surg. 56A:297–304; Green (1977) Clin. Orthop. 124:237–250; and Aston et al. (1986) J. Bone Joint Surg. 68B:29–35). During transplantation, the cell suspensions may be retained in the defect behind a piece of periosteal tissue that has been previously attached to the surface of the normal cartilage tissue. The rate of successful implantation using cell suspensions was found to be about 40%. It is believed that chondrocytes transplanted in this manner lose their viability during transplantation and that the procedure may result in the formation of fibrocartilage or islands of cartilage embedded in fibrous tissue at the site of the defect.
Three alternative approaches have been developed in an attempt to improve the success rate in treating mammalian articular cartilage defects. In the first approach, synthetic carrier matrices containing dispersed allogeneic chondrocytes may be implanted into the cartilage defect. The implanted chondrocytes hopefully produce and secrete components of the extracellular matrix thereby to form articular cartilage at the site of the defect in situ. In the second approach, synthetic carrier matrices containing chemotactic and mitogenic growth factors may be implanted into the cartilage defect. The growth factors hopefully induce the influx into, and the proliferation of chondrocyte progenitor cells within the matrix. The chondrocyte progenitor cells differentiate subsequently into chondrocytes that in turn secrete components of the extracellular matrix thereby to form articular cartilage at the site of the defect in situ. In the third approach, synthetic cartilage tissue may be grown in vitro and implanted subsequently into the cartilage defect.
In the first approach, the synthetic matrices or biological resorbable immobilization vehicles may be impregnated with allogeneic chondrocytes. A variety of synthetic carrier matrices have been used to date and include: three-dimensional collagen gels (U.S. Pat. No. 4,846,835; Nishimoto (1990) Med. J. Kinki University 15;75–86; Nixon et al. (1993) Am. J. Vet. Res. 54:349–356; Wakitam et al. (1989) J. Bone Joint Surg. 71B:74–80; Yasui (1989) J. Jpn. Ortho. Assoc. 63:529–538); reconstituted fibrinthrombin gels (U.S. Pat. No. 4,642,120; U.S. Pat. No. 5,053,050 and U.S. Pat. No. 4,904,259); synthetic polymer matrices containing polyanhydride, polyorthoester, polyglycolic acid and copolymers thereof (U.S. Pat. No. 5,041,138); and polyanhydride, polyorthoester hyaluronic acid-based polymers (Robinson et al. (1990) Calcif. Tissue Int. 46:246–253); and glycosaminoglycans or dextran sulfate.
The introduction of non-autologous materials into a patient, however, may stimulate an undesirable immune response directed against the implanted material. Such an immune response has been observed in rabbit models (Yoshinao (1990) J. Jpn. Orth. Assoc. 64:835–846. In addition, there is evidence to suggest that neo-cartilage may be formed around the periphery of the implant thereby preventing integration of the implant into the cartilage defect. See for example, Messner (1994) 40′″ Annual Meeting Orth. Res. Soc., New Orleans p. 239; and Nixon et al. (1994) 40′ Annual Meeting Orth. Res. Soc., New Orleans p. 241. Monitoring the formation and development of the resulting synthetic cartilage in situ can be difficult to perform and usually involves an arthroscopic or open joint examination. Furthermore, implants containing synthetic polymer components may be unsuitable for repairing large cartilage defects since polymer hydrolysis in situ may inhibit the formation of cartilage and/or its integration into the defect.
In the second approach, the defect may be filled with a biocompatible, biodegradable matrix containing growth factors to stimulate the influx of chondrocyte progenitor cells into the matrix in situ. The matrices optimally contain pores of sufficient dimensions to permit the influx into, and proliferation of the chondrocyte progenitor within the matrix. The matrix also may contain differentiating growth factors to stimulate the differentiation of chondrocyte progenitor cells into chondrocytes. The resulting chondrocytes hopefully secrete extracellular matrix components thereby to form cartilage at the site of the defect in situ. See for example, U.S. Pat. No. 5,206,023; U.S. Pat. No. 5,270,300; and EP 05 30 804 A1. This approach, however, may have problems similar to those associated with the first approach, herein above.
In the third approach, chondrocytes may be cultured in vitro to form synthetic cartilage-like material. The resulting cartilage may be implanted into the cartilage defect. This type of approach has the advantage over the previous methods in that the development of the synthetic cartilage material may be monitored prior to implantation. In addition, the resulting cartilage may be characterized biochemically and morphologically prior to implantation.
In vitro tissue engineering of cartilage on a polymer matrix is problematic because the resultant cell-polymer construct often has properties that are unfavorable for successful grafting. Particularly, the quantity and quality of secreted polymers does not adequately mimic that found in natural cartilage. Typically the amount of proteoglycan and collagen (measured relative to cell number or to total wet weight of the cell-polymer construct) is lower than in natural cartilage.
It has previously been demonstrated that hedgehog polypeptides (including hydrophobically-modified hedgehog polypeptides) have trophic effects on chondrocytes both in vivo and in vitro. (Iwasaki, Jikko & Le, 1999, Br. J Bone Joint Sur. 81, 1076; Iwanoto, et al., 1999, Crit Rev. Oral Biol. Med. 10, 477–86.). U.S. Pat. No. 5,972,385 suggests, but does not exemplify, the use of a oxidized polysaccharide matrix for the induction of connective tissue, including cartilage, which matrix is loaded with collagen and may be optionally loaded with a growth factor such as a hedgehog protein by co-valent linkage. The reference patent does not suggest for which type of connective tissue the use of a hedgehog protein is appropriate.
It would be advantageous to identify whether and in what optimal amounts of hedgehog polypeptides can be used to produce in vitro by culturing of chondrocytes in vitro a cartilage better adapted for implantation.